冷冻保存人类未成熟卵母细胞对其发育潜能的影响

目的 评价冷冻保存人类未成熟卵母细胞对其发育潜能的影响。方法 收集常规胞质内单精子显微注射-胚胎移植（ICSI-ET）中不成熟的卵母细胞168个,根据成熟程度分为生发小泡期（GV）卵母细胞103个和第1次减数分裂中期（MI）卵母细胞65个,再将各期卵母细胞分别分为冷冻组和对照组,冷冻组细胞经慢速冷冻-快速融解（慢冻-速融）后在体外培养成熟,对照组直接进行体外培养成熟。最后进行免疫荧光染色并观察。结果 GV冷冻组和GV对照组卵母细胞之间体外成熟率（分别为69．1％、74．3％）比较,差异无统计学意义（P〉0．05）;GV冷冻组和GV对照组卵母细胞之间纺锤体形态正常率（分别为28．9％、53．9％）和染色体形态正常率（分别为23．7％、50．0％）比较,差异有统计学意义（P〈0．05）;MI冷冻组和MI对照组卵母细胞之间体外成熟率（分别为68．6％、88．0％）比较,差异无统计学意义（P〉0．05）。MI冷冻组和MI对照组卵母细胞之间纺锤体形态正常率（分别为20．8％、54．6％）和染色体形态正常率（分别为25．0％、63．6％）比较,差异有统计学意义（P〈0．05）;GV冷冻组和MI冷冻组之间各项结果比较,差异均无统计学意义（P〉0．05）。结论 应用常规慢冻．速融方案并不能很好地保存未成熟卵母细胞的体外成熟后的纺锤体形成能力,未成熟的卵母细胞的发育潜能从而受损。

Spindle and chromosome configuration of human in vitro matured oocytes after slow freezing-fast thawing

OBJECTIVE: To evaluate the development of immature oocytes after freezing-thawing by conventional cryopreservation method for mature oocytes. METHODS: Immature oocytes were collected from stimulated ovaries of intracytoplasmic sperm injection (ICSI) cycles. Immature oocytes were in vitro matured directly or after slow freezing-fast thawing and immunostained for tubulin and chromatin and at last visualized by confocal microscopy. RESULTS: No statistical difference was found in maturity rate between freezing groups and the controls. There was a statistically significant increase in abnormalities of chromosome (23.7% vs. 50%) and spindle (28.9% vs. 53.9%) in the GV freezing group compared with the GV control (P < 0.05). MI groups gave the same results. But we did not find any statistical difference between GV frozen group and MI frozen group. CONCLUSIONS: Cryopreservation decreases the ability of immature oocytes to form normal meiosis spindle and induce abnormal division of chromosomes. This suggests that the routine slow freezing-fast thawing procedure for mature oocyte couldn't preserve the ability well to form normal meiosis spindle during in vitro maturation.